1. Field of the Invention
The invention is directed to discovery that the proapoptotic HIV-1-encoded protein Vpr induces mitochondrial membrane permeabilization via its physical and functional interaction with the mitochondrial inner membrane protein ANT (adenine nucleotide translocator, also called adenine nucleotide translocase or ADP/ATP carrier). HIV-1 Viral protein R (Vpr) interacts with the permeability transition pore complex (PTPC) to trigger ANT pore formation and/or mitochondrial membrane permeabilization (MMP) and consequent cell death (by apoptosis or any related mechanism of cell death).
2. Background
It is now recognized that mitochondria play an important role in controlling the life and death (the apoptosis) of cells (Kroemer and Reed 2000). Thus it seems that a growing number of molecules are involved in signal transduction, and that many metabolites (and certain viral effectors) act on the mitochondria and influence the permeabilization of mitochondrial membranes. Also, a certain number of experimental anti-cancer drugs kill cells by acting directly on mitochondrial membranes (Ravagnan et al., 1999; Larochette et al., 1999; Marchetti et al., 1999; Fulda et al., 1999; Belzacq et al., 2000). Therefore, the use of specific pro-apoptotic agents for mitochondria seems to be a concept that is emerging in anti-cancer chemotherapy (for reference: Costantini, et al., 2000). A possible outcome could be the use of cytoprotective molecules to treat illnesses associated with excess apoptosis (AIDS, neurodegenerative diseases, etc.) owing to their ability to stabilize mitochondrial membranes. Against this background, the identification (mode of action) of those molecular components that control the permeability of the mitochondrial membranes has become a major topic in biomedicine.
MMP is a key event of apoptotic cell death associated with the release of caspase activators and caspase-independent death effectors from the intermembrane space, dissipation of the inner transmembrane potential (ΔΨm), as well as a perturbation of oxidative phosphorylation G. Kroemer, N. Zamzami, S. A. Susin, Immunol. Today 18, 44–51 (1997). D. R. Green, J. C. Reed, Science 281, 1309–1312 (1998). J. J. Lemasters, et al., Biochim. Biophys. Acta 1366, 177–196 (1998). D. C. Wallace, Science 283, 1482–1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat. Cell Biol. 1, E209–E216 (1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes Dev. 13, 1988–1911 (1999). G. Kroemer, J. C. Reed, Nat. Med. 6, 513–519 (2000). Pro- and anti-apoptototic members of the Bcl-2 family regulate inner and outer MMP through interactions with the adenine nucleotide translocator (ANT; in the inner membrane, IM), the voltage-dependent anion channel (VDAC; in the outer membrane, OM), and/or through autonomous channel-forming activities G. Kroemer, N. Zamzami, S. A. Susin, Immunol. Today 18, 44–51 (1997). D. R. Green, J. C. Reed, Science 281, 1309–1312 (1998). J. J. Lemasters, et al., Biochim. Biophys. Acta 1366, 177–196 (1998). D. C. Wallace, Science 283, 1482–1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat. Cell Biol. 1, E209–E216 (1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes Dev. 13, 1988–1911 (1999). G. Kroemer, J. C. Reed, Nat. Med. 6, 513519 (2000). I. Marzo, et al., Science 281, 2027–2031 (1998). S. Shimizu, M. Narita, Y. Tsujimoto, Nature 399, 483–487 (1999). S. Shimizu, A. Konishi, T. Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA 97, 3100–3105 (2000). S. Desagher, et al., J Cell Biol. 144, 891–901 (1999).
ANT and VDAC are major components of the permeability transition pore complex (PTPC), a polyprotein structure organized at sites at which the two mitochondrial membranes are apposed. G. Kroemer, N. Zamzami, S. A. Susin, Immunol. Today 18, 44–51 (1997). D. R. Green, J. C. Reed, Science 281, 1309–1312 (1998). J. J. Lemasters, et al., Biochim. Biophys. Acta 1366, 177–196 (1998). D. C. Wallace, Science 283, 1482–1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat. Cell Biol. 1, E209–E216 (1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes Dev. 13, 1988–1911 (1999). G. Kroemer, J. Reed, Nat. Med. 6, 513–519 (2000). M. Crompton, Biochem. J. 341, 233–249 (1999).
The adenine nucleotide translocator (ANT) plays an important role in the process that triggers the permeabilization of mitochondrial membranes, and subsequent apoptosis (Marzo, et al., 1998; Brenner, et al., 2000). In the cellular context, ANT is inserted into the internal membrane of mitochondria and has two opposing functions. On the one hand, ANT is a vital antiport for cellular bioenergetics and is specific to ATP and ADP. On the other hand, ANT can form a non-specific lethal pore through the action of certain ligands (natural or xenobiotic) that eliminate the mitochondrial electrochemical gradient.
The HIV-1 regulatory protein Vpr has pleiotropic effects on viral replication and cellular proliferation, differentiation, cytokine production, and NF-κB-mediated transcription. M. Emerman, M. H. Malim, Science 280, 1880–1884 (1998). A. D. Frankel, J. A. T. Young, Annu. Rev. Biochem. 67, 1–25 (1998). M. Bukrinsky, A. Adzhubei, J. Med. Virol. 9, 39–49 (1999). In addition, Vpr can localize to mitochondria. I. G. Macreadie, et al., Proc. Natl. Acad. Sci. USA 92, 2770–2774 (1995). I. G. Macreadie, et al., FEBS Lett. 410, 145–149 (1997). K. Muthami, L. J. Montaner, V. Ayyavoo, D. B. Weine, DNA and Cell Biology 19, 179–188 (2000). E. Jacotot, et al., J. Exp. Med. 191, 33–45 (2000). Full length (Vpr1–96) or truncated synthetic forms of Vpr act on the PTPC to induce all mitochondrial hallmarks of apoptosis, including ΔΨm loss and the release of cytochrome c and apoptosis inducing factor (AIF). E. Jacotot, et al., J. Exp. Med. 191, 33–45 (2000). The MMP-inducing activity of Vpr resides in its C-terminal moiety (Vpr52–96), within an a-helical motif of 12 amino acids (Vpr71–82) containing several critical arginine (R) residues (R73, R77, R80) which are strongly conserved among different pathogenic HIV-1 isolates. L G. Macreadie, et al., Proc. Natl. Acad. Sci. USA 92, 2770–2774 (1995). I. G. Macreadie, et al., FEBS Lett. 410, 145–149 (1997). E. Jacotot, et al., J. Exp. Med. 191, 33–45 (2000).
Depending on the apoptotic stimulus, permeabilization may affect the OM and IM in a variable fashion and may or may be not accompanied by matrix swelling. G. Kroemer, N. Zamzami, S. A. Susin, Immunol. Today 18, 44–51 (1997). D. R. Green, J. C. Reed, Science 281, 1309–1312 (1998). J. J. Lemasters, et al., Biochim. Biophys. Acta 1366, 177–196 (1998). D. C. Wallace, Science 283, 1482–1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat. Cell Biol. 1, E209–E216 (1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes Dev. 13, 1988–1911 (1999). G. Kroemer, J. C. Reed, Nat. Med. 6, 513–519 (2000). In vitro experiments performed on purified mitochondria or proteins reconstituted into artificial membranes suggest at least two competing models of MMP. On the one hand, pore formation by ANT has been proposed to account for IM permeabilization, osmotic matrix swelling, and consequent OM rupture, resulting because the surface area of the IM with its folded christae exceeds that of the OM. In support of this hypothesis, proapoptotic molecules such as Bax, atractyloside, Ca2+, and thiol oxidants cause ANT (which normally is a strictly specific ADP/ATP antiporter) to form a non-specific pore (I. Marzo, et al., Science 281, 2027–2031 (1998); N. Brustovetsky, M. Klingenberg, Biochemistry 35, 8483–8488 (1996); C. Brenner, et al., Oncogene 19, 329–336 (2000)). On the other hand, VDAC has been suggested to account for a primary OM permeabilization not affecting IM (S. Shimizu, M. Narita, Y. Tsujimoto, Nature 399, 483–487 (1999). S. Shimizu, A. Konishi, T. Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA 97, 3100–3105 (2000)). In favor of this hypothesis, the permeabilization of VDAC-containing liposomes to sucrose or cytochrome c is enhanced by Bax and inhibited by Bcl-2 in vitro. S. Shimizu, M. Narita, Y. Tsujimoto, Nature 399, 483–487 (1999). S. Shimizu, A. Konishi, T. Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA 97, 3100–3105 (2000).
Recent studies have revealed the existence of several viral apoptosis inhibitors acting on mitochondria. For example, adenovirus, Epstein Barr virus, Herpes virus saimiri, and Kaposi sarcoma-associated human herpes virus 8 produce apoptosis-suppressive Bcl-2 homologs. E. H.-Y. Cheng, et al., Proc. Natal. Aced. Sic. USA 94, 690–694 (1997). J. H. Than, D. Mocha, E. White, Oncogene 17, 2993–3005 (1998). T. Degauss, et al., J. Viral. 72, 5897–5904 (1998). W. L. Marshall, et al., J. Virol. 73, 5181–5185 (1999). In addition, several viruses encode PTPC-interacting proteins without any obvious homology to the Bcl-2/Bax family. The cytomegalovirus apoptosis inhibitor pUL37x (V. S. Goldmacher, et al., Proc. Natl. Acad Sci. USA 96, 12536–12541 (1999).) and Vpr, an HIV-1-encoded apoptosis inducer, selectively bind to ANT. The proapoptotic p13 (II) protein derived from the X-II ORF of HTLV-1 is also targeted to mitochondria via a peptide motif that bears structural similarities to the mitochondriotoxic domain of Vpr. V. Ciminale, et al., Oncogene 18, 4505–4514 (1999). Moreover, the pro-apoptotic, MMP-inducing hepatitis virus B protein X interacts with VDAC. Z. Rahmani, K. W. Huh, R. Lasher, A. Siddiqui, J. Virol. 74, 2840–2846 (2000). Thus, both VDAC and ANT emerge as major targets of viral apoptosis regulation and, perhaps, as targets for pharmacological intervention on viral pathogenesis and/or other pathologies linked to apoptosis dysregulations (i.e., cancer, ischemia, neurodegenerative diseases, etc.). Apoptosis is a process that develops in several phases: (1) an initiation phase, which is extremely heterogeneous and during which the biochemical pathways participating in the process depend on the apoptosis-inducing agent; (2) a decision phase, which is common to different types of apoptosis, during which the cell “decides” to commit suicide; and (3) a common degradation phase, which is characterized by the activation of catabolic hydrolases (caspases and nucleases). Although the activation of caspases (cysteine proteases cleaving at aspartic acid [Asp] residues) and nucleases is necessary for the acquisitions of the full apoptotic morphology, it appears clear that inhibition of such enzymes does not inhibit cell death induced by a number of different triggers: Bax, Bak, c-Myo, PML, FADD, glucocorticoid receptor occupancy, tumor necrosis factor, growth factor withdrawal, CXCR4 cross-linking, and chemotherapeutic agents, such as etoposide, camptothecin, or cisplatin. In the absence of caspase activation, cells manifest a retarded cytolysis without characteristics of advanced apoptosis, such as total chromatin condensation, oligonucleosomal DNA fragmentation, and formation of apoptotic bodies. However, before cells lyse, they do manifest a permeabilization of both mitochondrial membranes with dissipation of the inner transmembrane potential (Δψm) and/or the release of apoptogenic proteins, such as cytochrome c and apoptosis-inducing factor (AIF) via the outer membrane. These results have invalidated the hypothesis that caspase activation is always required for apoptotic cell death to occur. Rather, cell death is intimately associated with the permeabilization of mitochondrial membranes.
The understanding of apoptosis has recently been facilitated by the development of cell-free systems. Instead of considering the cell as a black box, subcellular fractions (e.g., mitochrondria, nuclei, and cytosol) are mixed together with the aim to reconstitute the apoptosis phenomenon by recapitulating the essential steps of the process in vitro. It appears that proapoptotic second messengers, whose nature depends on the apoptosis-inducing agent, accumulate in the cytosol during the initiation phase. These agents then induce mitochondrial membrane permeabilization, allowing cells to enter the decision phase. The apoptotic changes of mitochondria consist in a Δψm loss, transient swelling of the mitochondrial matrix, mechanical rupture of the outer membrane and/or its nonspecific permeabilization by giant protein-permanent pores, and release of soluble intermembrane proteins (SIMPs) through the outer membrane. Once the mitochondrial membrane barrier function is lost, several factors, e.g., the metabolic consequences at the bioenergetic level, the loss of redox homeostasis, and the perturbation of ion homeostasis, contribute to cell death. The activation of proteases (caspases) and nucleases by SIMP's is necessary for the acquisition of apoptotic morphology. This latter phase corresponds to the degradation step, beyond the point of no return of the apoptotic process. Different SIMPs provide a molecular link between mitochondrial membrane permeabilization and the activation of catabolic hydrolases: cytochrome c (a heme protein that participates in caspase activation), certain procaspases (in particular, procaspases 2 and 9, which in some cell types, are selectively enriched in mitochondria), and AIF. AIF is a nuclear-encoded intermembrane flavoprotein that translocates to the nucleus where it induces the caspase-independent peripheral chromatin condensation and the degradation of DNA into 50-kilobase pair fragments.
The mechanism of mitochondrial membrane permeabilization is not completely understood. Some investigators prefer the hypothesis that proapoptotic members of the Bcl-2 family are inserted in the outer membrane where they oligomerize and form cytochrome c permeant pores in an autonomous fashion, not requiring the interaction with other mitochondrial membrane proteins. However, Bax-induced membrane permeabilization is inhibited by cyclosporin A (CsA) and bongkrekic acid (BA), two inhibitors of formation of the permeability transition pore (or “megachannel”), suggesting that sessile mitochrondrial proteins (the targets of CsA and BA) are involved in this process. The permeability transition pore has a polyprotein structure that is formed at the contact sites between the inner and outer membranes. One of the key proteins of the permeability transition pore complex (PTPC) is the adenine nucleotide translocator (ANT). ANT, the target of BA, is the most abundant inner membrane protein, ANT normally functions as a specific carrier protein for the exchange of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), but it can become a nonspecific pore.
An interesting property of the PTPC is that the permeabilization of the inner and/or outer mitochondrial membranes compromises the bioenergetic equilibrium of the cell (e.g., it provokes the oxidation of reduced NADPH and glutathione, the depletion of ATP, and the dissipation of Δψm and effects the homeostasis of intracellular ions (e.g., by releasing Ca2+ from the matrix). Intriguingly, all of these changes themselves increase the probability of PTPCs opening. This has two important implications. First, the consequences of PTPC opening themselves favor opening of the PTPC in a self-amplification loop that coordinates the lethal response among mitochondria within the same cells. Second, this implies that the final result of PTPC opening is a stereotyped ensemble of biochemical alterations, which does not depend on the initiating stimulus, be it a specific proapoptotic signal transduction cascade or nonspecific damage at the energy or redox levels.
Chemotherapy aims at the specific eradication of cancer cells, mostly through the induction of apoptosis. Gene therapy can employ Bax-delivering vectors, thereby indirectly targeting mitochondria to induce apoptosis. In contrast to such proteins, certain peptides readily penetrate the plasma membrane and thus can be used as true pharmacologic agents. Mastoparan, a peptide isolated from wasp venom, is the first peptide known to induce mitochondrial membrane permeabilization via a CsA-inhibitable mechanism and to induce apoptosis via a mitochondrial effect when added to intact cells. This peptide has an α-helical structure and possesses some positive charges that are distributed on one side of the helix. A similar peptide (KLAKLAKKLAKLAK or (KLAKLAK)2 (K=lysine, L=amine, and A=leucine) has been found recently to disrupt mitochondrial membranes when it is added to purified mitochondria, although the mechanisms of this effect have not been elucidated. (Ellerby, H. M. et al., Anti-cancer activity of targeted pro-apoptotic peptides, Nature Med. 5, 1032–1038 (1999)).
The proapoptotic 96 amino acid protein viral protein R (Vpr) from human immunodeficiency virus-I contains a comparable structural motif (aa 71–82), i.e., an α-helix with several cationic charges that concentrate on the same side of the helix. Vpr, as well as Vpr derivatives containing this “mitochondriotoxic” domain cause a rapid CsA and BA-inhibited dissipation of the Δψm as well as the mitochondrial release of apoptogenic proteins, such as cytochrome c or AIF. The same structural motifs relevant for cell killing appear to be responsible for the mitochondriotoxic effects of Vpr. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the ANT combined with Bax, but this effect is prevented by the addition of recombinant Bcl-2. According to surface plasmon resonance studies, the Vpr C-terminus binds purified ANT with a high affinity in the nanomolar range. E. Jacotot et al., J. Exp. Med. 191, 33–45 (2000), which is specifically incorporated herein by reference. In addition, a biotinylated Vpr-derived peptide (Vpr52–96) may be employed as bait to specifically purify a mitochondrial molecular complex containing ANT and the VDAC. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than are control cells. Thus, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC. In analogy to Vpr, the p13 (II) protein derived from the X-II open reading frame of HTLV-1 is targeted to mitochondria and can cause a dissipation of the Δψm and mitochondrial swelling. Mitochondrial targeting of this protein has been mapped to a decapeptide sequence that contains several Arg residues that are asymmetrically distributed in the α-helix. However, Arg-Ala substitutions within the mitochondriotoxic domain of p13 (II) did not abolish the mitochondrial targeting of p13.
Lethal peptides may be targeted to mitochondria and more specifically, at least in the case of Vpr, to the PTPC. Ellerby et al. recently have fused the mitochondriotoxic (KLAKLAK)2 motif to a targeting peptide that interacts with endothelial cells. Such a fusion peptide is internalized and induces mitochondrial membrane permeabilization in angiogenic endothelial cells and kills MDA-MD-435 breast cancer xenografts transplanted into nude mice. Similarly, a recombinant chimeric protein containing interleukin 2 (IL-2) protein fused to Bax selectively binds to and kills IL-2 receptor-bearing cells in vitro. Thus, specific cytotoxic agents that target surface receptors, translocate into the cytoplasm, and induce apoptosis via mitochondrial membrane permeabilization might be useful in treating cancer.
A recurrent problem with conventional chemotherapeutic agents is that they exploit endogenous apoptosis-induction pathways that may be compromised by alterations such as mutations of p53, increased antioxidant activity, blockade of the CD95/CD95L pathway, overexpression of Bcl-2-like proteins, etc. One possible strategy to enforce cell death is to trigger downstream events of the common apoptotic pathway. Thus, adenovirus-mediated transfer of caspases has been proposed as one strategy to induce cell death beyond any regulation. An alternative strategy is to use mitochondriotoxic agents that induce cell death irrespective of the upstream control mechanisms and irrespective of the status of caspases and endogenous caspase inhibitors. As an example, LND, arsenite, or CD437 induce cell death independently of the p53 status via a pathway that is not affected by caspase inhibitors. Similarly, betulinic acid and Vpr trigger CD95 (Apo-1/Fas)- and p53-independent apoptosis, and both permeabilize mitochondrial membranes in a caspase-independent fashion. As a result, these types of agents may prove to be highly useful in killing normally resistant cells. Moreover, the future of tumor therapy may profit from the design of agents that overcome the Bcl-2-mediated stabilization of mitochondrial membranes as well as from targeting amphipathic peptides or peptidomimetics to defined cellular populations or tissues.
Selective eradication of transformed cells by use of mitochondrion-specific agents should be effective. One strategy is to target a toxic agent to selected cell types on the basis of the specific expression of surface receptors. Another, yet to be developed, strategy would aim at exploiting difference in the composition or regulation of the PTPC between normal and tumor cells. Future research will tell to which extent cell targeting (by use of retroviral or adenoviral vectors, use of integrin-specific domains, etc.) and/or targeting of tumor-specific alterations in the PTPC will prove to be useful in cancer therapy, and also in the treatment of neurodegenerative diseases hypothetically linked to mitochondrial dysfunction (i.e., Friedrich ataxia, Hereditary spastic paraplegia, Huntington disease, Amyotrophic lateral sclerosis, Parkinson disease, Alzheimer disease) and treatment of acute organ failure that may involve regulatory events acting at the level of MMP (i.e., ischemia) (Kroemer, G. et al., Mitochondrial control of cell death, Nature Med., vol. 6, no. 5, 513–519 (1999)).
Thus, there exists a need in the art for methods and reagents for regulating mitochondrial permeabilization and apoptosis.